Nitrogen is an essential element for plant growth. A number of plants such as the legumes (soybean, peas, beans, alfalfa, clover, peanut, black locust, etc.) and various woody angiosperms (alder, casuarina, etc.) are able to provide their nitrogen requirements by forming a symbiotic association with certain soil bacteria. The bacteria live within root (or in some species, stem) structures called nodules where they reduce atmospheric nitrogen gas (N.sub.2) to a form of nitrogen that the plant can use in the production of protein, DNA, etc. Therefore, these plants do not require expensive and environmentally harmful nitrogenous fertilizers to support their growth and provide commercially acceptable yields. Nitrogenase is the bacterial enzyme responsible for nitrogen fixation. However, not all of nitrogenase activity is associated with N.sub.2 fixation, since a portion (usually 25-40%) of the activity involves hydrogen gas production.
Biological nitrogen fixation will play an increasingly important role in future agricultural practice. However, the process is very sensitive to several environmental factors such as drought stress or the presence of nitrogenous fertilizer in the soil. In these situations, studies with legumes have shown that the nodule restricts oxygen diffusion to the bacteria and consequently nitrogenase activity declines due to severe oxygen limitation. In a field situation, it is very difficult to determine whether the nodules are actively fixing nitrogen gas and therefore difficult to know whether or not to take remedial action. Since most legume nodules are very sensitive to disturbance, it is not possible to dig up the root system in order to determine nodule activity by any of the known methods. Known methods for measuring symbiotic nitrogen fixation include:
(a) The plant nitrogen increment method in which plants are harvested at different times and their nitrogen content is measured. This method is time consuming, destructive and does not distinguish between N.sub.2 fixation and fertilizer nitrogen uptake. It also requires an expensive chemical assay, and when the result is known it is generally too late to take any required action to increase nitrogen input into the crop.
(b) Isotopic methods in which the .sup.15 N and .sup.14 N content of plant tissues are measured. Due to differences between combined nitrogen (NO.sub.3.sup.-, NH.sub.4.sup.+) assimilation and N.sub.2 fixation in the assimilation of .sup.15 N and .sup.14 N- containing molecules, it is possible to estimate rates of N.sub.2 fixation from a knowledge of the isotopic composition of the plant, soil and atmospheric N pools. Alternatively, enriched levels of .sup.15 NO.sub.3.sup.- or .sup.15 N.sub.2 can be provided to the plant and the contribution of each N source can be measured over a defined interval of time. These isotopic methods are destructive, time consuming, require expensive isotopes and analytical instrumentation and by the time the result is obtained, it would likely be too late to take remedial action for the crop.
(c) The acetylene reduction assay method in which the plant roots and nodules are exposed to 10% acetylene, and the production of ethylene is measured over time. The N.sub.2 fixing enzyme, nitrogenase, uses acetylene as an alternative substrate and reduces it to ethylene. While the method is quick, relatively inexpensive and, in theory, measures nitrogenase activity directly, it is notorious for producing artifactual results. Also expensive equipment is needed and it is usually destructive to the plant.
(d) Monitoring hydrogen gas production, a by-product of nitrogenase activity, from nodulated roots in air and Ar:O.sub.2 gas. While the method is non-invasive and relatively inexpensive, not all symbiosis evolve the hydrogen gas that is produced by the nitrogenase enzyme. Also, the method would be difficult to use in field studies.
It will be apparent that none of the above techniques are of much use to Plant Breeders who want to screen large numbers of plants for maximal nitrogenase activity under normal field conditions. Neither are they of use to farmers who need to know whether their N.sub.2 -fixing crops are actively fixing the nitrogen they will need for optimal growth and yield, and therefore whether or not they should irrigate and/or apply chemical fertilizer. What is needed, therefore, is a rapid, non-invasive technique to obtain a reasonably accurate estimate of nitrogen fixation in plants actively growing in the field.
Recent studies with legumes have shown that oxygen plays a critical role in regulating root nodule metabolism and nitrogenase activity in legumes. The bacteria in the root nodules require large amounts of oxygen for respiration, yet oxygen is a potent irreversible inhibitor of the nitrogenase enzyme. Hence oxygen in the bacteria-infected cells must be maintained at a very low level. The nodule does this by regulating its permeability to O.sub.2 diffusion from the soil environment into the bacteria-infected cells. The O.sub.2 concentration is maintained at such a low level that it limits the supply of respiratory energy available for nitrogenase activity. Thus there is, under a wide range of environmental and physiological conditions, a strong correlation between infected cell oxygen concentration and nitrogenase activity. Nodule respiration and nodule permeability are also correlated with nitrogenase activity under many environmental and physiological conditions.
The oxygen concentration in the infected cells is too low to be measured by oxygen electrodes or by mass spectrometry, so the rapid measurement of oxygen concentration cannot be effected directly. However, the infected cells of legume nodules contain a high concentration of a red colored, myoglobin-like compound called leghemoglobin, which reversibly binds oxygen and acts to facilitate the diffusion of oxygen to the bacteria. When oxygen binds to leghemoglobin, it causes a change in the leghemoglobin absorption spectrum, and this change can be used as the basis for the spectrophotometric determination of fractional oxygenation of leghemoglobin. From the measurement of fractional leghemoglobin oxygenation, and a knowledge of the rate constants for leghemoglobin oxygenation and deoxygenation, an estimate of the free O.sub.2 concentration in the infected cells can be calculated. Many non-leguminous N.sub.2 fixing plants are also known to contain hemoglobins, and it should be possible to use the methodology described herein to measure the oxygen concentration in these nodules.
Hemoglobin oxygen saturation has been studied in mammalian systems for many years and there are several instruments, known as oximeters, available to measure non-invasively the proportion of hemoglobin oxygen saturation in blood. One such system is described in some detail in IEEE Trans. Biomed. Eng. 35: 185-197 (1988). However, these oximeters, which will be discussed in more detail hereinafter, are sensitive to ambient light, not designed for use with small nodules and therefore they are not suitable for agricultural field use.